This invention relates to a DNA expression vector for the production of a hybrid polypeptide and particularly to a recombinant hybrid polypeptide composed of a polypeptide of interest fused to a polypeptide that contains a domain or recognition sequence for attachment of the prosthetic group biotin. More particularly, this invention relates to a hybrid polypeptide wherein the biotinylated hybrid polypeptide binds to avidin. This invention also describes a process for producing this hybrid polypeptide in a procaryotic or a eucaryotic protein expression system. Such a hybrid polypeptide can be obtained in high purity and yield using avidin monomer affinity chromatography (U.S. patent application Ser. No. 07/414,785, filed Sep. 29, 1989 and entitled HPLC AVIDIN MONOMER AFFINITY RESIN now U.S Pat. No. 5,276,062). U.S. Pat. No. 5,276,062 discloses a monomeric avidin polypeptide ligand and a novel and particularly efficacious process for isolating synthetic or natural molecules and/or biotinylated derivatives thereof, by adsorbtion of the molecules of interest onto a novel affinity media which contains avidin fixed to a solid inert support. The disclosure of Ser. No. 07/414,785 is incorporated herein by reference.
Synthesis of commercially important peptides and proteins has been limited by high production and purification costs and poor product recovery. Until recently, animals, microorganisms, plants, cadavers, serum, and urine have been the only sources from which bioactive polypeptides could be purified.
Biological synthesis of valuable polypeptides has been made possible in commercial quantities through advances in recombinant DNA technology. Recombinant DNA molecules directing the synthesis of commercially useful polypeptides can be introduced into procaryotic or eucaryotic expression systems. For example, recombinant DNA technology has enabled human growth hormone production by recombinant bacteria, and fermentation now replaces the traditional source. To date, biological synthesis is the only practical approach to the commercial-scale synthesis of peptides of greater than 20 amino acid residues.
Once synthesized, the desired polypeptide product must be purified from a complex mixture of cellular components. The degree of purification depends upon the intended application of the polypeptide. The cost of purification can account for up to 70% of the cost of production, as substantial losses of active ingredient usually occur during multistep purification processes.
Polypeptide purifications are usually achieved through one or more processes which are based upon physical properties of the polypeptide of interest. For example, proteins may be separated on the basis of solubility, size, ionic properties or affinity for specific ligands; usually several of these techniques are required to achieve acceptable purity.
Affinity resin chromatography can greatly reduce the number of purification steps required to achieve the desired level of purity. Affinity purification is based upon a specific binding interaction between a polypeptide to be purified and a ligand which is usually attached to a solid support. As used herein, the polypeptide binds to the ligand by virtue of a prosthetic group bound to an attachment domain present on the polypeptide. When a complex mixture such as a cell extract or crude mixture of synthetic peptides is passed over an affinity resin, the polypeptide to be purified is selectively retained by the resin and all molecules lacking the prosthetic group on the attachment domain are washed away from the resin. Therefore, in a single step, the polypeptide of interest may be recovered in high purity.
In order to use affinity chromatography to advantage for polypeptide purification, recombinant DNA technology can be used to construct chimeric gene fusions for recombinant hybrid polypeptides which in bacterial host cells incorporate the following elements: a 5' promoter; DNA coding for a polypeptide of interest; DNA coding for a polypeptide that contains a ligand binding domain; and optionally ribosomal terminators, such as the rrnB terminators found on the E. coli expression vector pkk223-3 (Brosius, J. and Holy A., Proc Nat Acad Sci USA 81:6929-6933 (1984); Brosius, J. et al Plasmid 6:112-118 (1984)). Suitable promoters are those which maximize expression of the desired gene in the host cell, and factors to be considered in promoter construction are discussed by Old and Primrose in Chapter 7 of Principles of Gene Manipulation 3rd Edition (Blackwell Scientific Publications, Palo Alto Calif. 1985). Examples of bacterial promoters appropriate for expression of cloned genes include the P.sub.L, tac, lac, and trp promoters (ibid). The DNA used to construct chimeric gene fusions can be obtained from organisms or can be novel synthetic DNA fragments, or combinations thereof. The DNA sequences are assembled into a chimeric gene, which is inserted into a DNA expression vector in such a manner that in the appropriate host organism, the polypeptide of interest and the polypeptide for attachment to the affinity resin are produced as a single polypeptide chain.
In the present invention, a binding domain, or recognition sequence directs the attachment of a prosthetic group, such as biotin, to the hybrid polypeptide. The biotinylated hybrid polypeptide can be specifically selected by affinity ligand compositions such as the avidin monomer resin (U.S. patent application Ser. No. 414,785). A single purification step can therefore separate the protein of interest from complex mixtures, such as crude bacterial lysates, with high levels of recovery in a single chromatographic step, thus alleviating the recovery problems inherent to multistep purification processes. Such a combination of hybrid polypeptide and avidin monomer affinity resin would confer significant advantages to the purification of commercially useful polypeptides over existing processes.
Other systems for affinity purification of hybrid recombinant polypeptides have been described. However, significant technical obstacles limit their use for commercial-scale polypeptide purification. For example, chimeric genes encoding polypeptides containing a polyarginine C-terminal tail (Sassenfeld and Brewer, Biotechnology 2:76-81 (1984)) or polyhistidine domain (Smith et al. J. Biol Chem 263:7211 (1988)) can facilitate separation by ion exchange or metal chelate ion chromatography. Such systems are not broadly applicable because affinity interaction depends upon physical properties of the fusion polypeptide (chargeability to chelate metals), and it is not always possible to achieve sufficient change in these physical properties to permit affinity binding.
Another type of affinity chromatography is immunoaffinity chromatography, wherein polypeptides of interest are fused to immunogenic proteins such as E. coli beta-galactosidase (Ruther and Muller-Hill, EMBO J 2:1791-1794 (1983)) or small hydrophilic peptides (Hopp et al., U.S. Pat. No. 4,703,004 (1988)) to achieve purification. Polypeptides fused to staphylococcal protein A can be purified using IgG-Sepharose (Nilsson et al. EMBO J. 4:1075-1080 (1985), Lowenadler et al. EMBO J. 5:2393-2398 (1986)). Polypeptides fused to Protein G can be isolated using albumin as the immobilized ligand (Nygren et al. J. Mol. Recognition, 1:69 (1988)). A critical disadvantage limiting the usefulness of these methods is that extreme conditions, including the use of denaturants, are necessary to remove the fusion proteins from the affinity resin, which may destroy biological activity if native folding cannot be achieved. Low product recovery rates can also limit the usefulness of such systems.
Affinity based upon the binding of small molecules by a large protein is known as substrate-affinity chromatography. A small molecule, a ligand, forms a complex with a specific ligate. Examples of ligand:ligate combinations include avidin:biotin (Green in Advances in Protein Chemistry Vol 29 pp 85-133 Anson et al., Eds. (1975)), streptavidin:biotin (PCT/US85/01901, Meade and Garvin (1985)), lipoic acid:avidin (Green ibid), chloramphenicol acetyl transferase:acetyl CoA (EPO 0131363, Bennet et al. (1984)), beta-galactosidase: para-aminophenyl-beta-D-thio-galactoside (Offensberger et al. Proc. Natl Acad. Sci USA 82:7540-7544 (1985)), phosphate binding protein:hydroxyapatite (Anba et al. Gene 53:219 (1987)), maltose binding protein:starch (EPO 286239, Guan et al. 1988), and glutathione S-transferase:glutathione (Smith and Johnson Gene 67:21-30 (1988)).
In recent years, the unique properties of the prosthetic group biotin and its exceptionally high affinity (10.sup.15 M.sup.-1) and specificity for the proteins avidin and streptavidin (Green ibid.) have been exploited to devise powerful and widely applicable tools for microbiology, biochemistry and medical science (Wilchek and Bayer Analyt Biochem 171:1-32 (1988), Bayer and Wilchek Methods in Biochem Anal 26:1-45 (1980)).
Biotin (Ann N.Y. Acad. Sci 447:1-441, Dakshinamurti and Bhagavan, Eds. (1985)) is a prosthetic group found on only a few protein species. Attachment in vivo is mediated by biotin holoenzyme synthetases which recognizes a highly conserved attachment domain and catalyzes the covalent attachment of biotin to that domain (Wood et al, J Biol Chem 225:7397-7409 (1980); Shenoy and Wood, FASCB S 2:2396-2401 (1988)). Experiments using recombinant DNA technology have shown that biotin holoenzyme synthetases will biotinylate heterologous polypeptides containing this conserved attachment domain. For example, the 1.3S subunit of the enzyme transcarboxylase from Propionibacterium, which contains the conserved sequence, when cloned and expressed in E. coli is biotinylated by the E. coli synthetase (Murtif et al. Proc Nat Acad Sci USA 82:5617-5621 (1985)).
A polypeptide or part of a polypeptide containing the conserved biotin attachment domain, such as entire 1.3S (SEQ ID NO:1) protein or the biotin-binding recognition sequence identified within the 1.3S protein from Propionibacterium, (SEQ ID NO:2) can be incorporated into a hybrid recombinant polypeptide. Such a hybrid polypeptide containing a biotin attachment domain fused to one or more polypeptides of interest could be used to achieve the separation of virtually any recombinant protein based upon the affinity of the ligand avidin for the ligate biotin.
Avidin:biotin chromatography shares advantages generally applicable to substrate affinity chromatography systems for commercial-scale polypeptide purification. Substrate-affinity resins are generally inexpensive. Fusion proteins can be recovered using mild conditions by elution with free ligand. Post-translational addition of the biotin prosthetic group is independent of the final folded state of the protein (Wood et al. J Biol Chem 255:7397-7409 (1980)), an advantage when the host cell performs no post-translational modifications on the recombinant polypeptide. A ligand domain such as the domain directing biotin attachment would be particularly advantageous for recovery of fusion proteins found in inclusion bodies or for recovery of insoluble proteins which require denaturants or zwitterionic detergents for solubilization during extraction, prior to affinity chromatography.
Cronan (J Biol Chem 265:10327-10333 (1990)) has used a recombinant DNA plasmid from E. coli (Murtif et al. Proc Nat Acad Sci USA 82:5617-5621 (1985)) to construct fusion genes containing segments of the 1.3S gene, which contain the biotin attachment domain. Cronan (ibid) demonstrated that 1.3S sequences can be used to specifically label proteins in vivo, and to purify proteins from crude cell lysates by avidin affinity chromatography.
Cronan's (ibid) chimeric genes were constructed by fusing the 3' end of the genes of interest to the 5' end of the 1.3S gene, yielding hybrid recombinant polypeptide having the polypeptides of interest fused to the N-terminus of the 1.3S polypeptide. Such fusions are consistent with the teachings of Murtif and Samols (J Biol Chem 262:11813-11816 (1987)) who teach the fusion of the 3' end of the gene of interest to the 5' end of the 1.3S gene (the N-terminus of the 1.3S polypeptide) to avoid interfering with the attachment of biotin to its binding domain. Murtif and Samols (ibid) teach that the conformation of the COOH terminus of the 1.3 S polypeptide, and the spatial relationship between this region and a lysine residue positioned exactly 35 residues from the COOH terminus position to which biotin is attached in vivo, are essential for proper enzymatic recognition and biotinylation of the 1.3S polypeptide. Murtif and Samols (Ibid) further teach that the conformation of the carboxyl terminal region of the 1.3S polypeptide is critical for biotinylation, and that altering the hydrophobicity of the carboxyl terminal region of the 1.3S polypeptide "eliminates biotinylation." Murtif Samols did observe biotinylation of 1.3S polypeptides, each lengthened by two amino acids at the 1.3S carboxyl terminus. However, such additions of two amino acids to the C-terminus did not substantially change its hydrophobicity and such small additions would not be expected to change the conformation of the C-terminus. Therefore, Murtif and Samols (ibid) and Cronan (ibid) teach away from fusing the polypeptide of interest to the COOH terminus of the 1.3S polypeptides or fragments of the 1.3S polypeptide, so that the correct conformation of the biotin attachment region may be preserved.
It was therefore surprising to find that if one went against the teachings of Murtif and Samols (ibid) and Cronan (ibid) and fused a polypeptide of interest to the C-terminus of the 1.3S polypeptide, that the appropriate lysine residue of the 1.3S polypeptide within the hybrid was indeed biotinylated, since the addition of a polypeptide substantially longer than two amino acid residues would be expected from earlier teachings of Murtif and Samols to alter the conformation of the C terminus of the 1.3S polypeptide and preclude biotinylation. Additionally, hybrid polypeptides in which the 1.3S polypeptide was fused at its C-terminus to the polypeptide of interest were recovered in high purity and high yield in a single chromatographic step using Avidin Monomer Affinity chromatography (U.S. application Ser. No. 07/414,785) now U.S. Pat. No. 5,276,062.
Further, an advantage is derived from fusing the polypeptide of interest to the C-terminus of the 1.3S polypeptide and not to its N-terminus. Protein expression level in a host cell is determined by a number of factors, including promoter strength and optimal initation of protein translation (ibid). Promoter strength contributes to the efficiency of transcription of messenger RNA. Optimization of the processes involved in the initiation of translation is important to achieving high levels of protein expression in the host cell. When polypeptides of interest are introduced at the 3' terminus of the 1.3S gene, no change is made to the optimal placement of the 5' terminus of the 1.3S gene directly adjacent to the promoter and 5' regulatory sequences. Insertion of the polypeptides of interest between the promoter and 5' terminus of the 1.3S gene may require additional expermination to achieve maximal expression levels in the host cell.
It is an object of this invention to provide a recombinant hybrid polypeptide comprising a polypeptide of interest fused to an avidin-binding polypeptide containing a domain for attachment of biotin, with that polypeptide of interest being fused to the avidin-binding polypeptide at the C-terminus of the avidin-binding polypeptide.
It is a further object of this invention to provide a DNA expression vector containing DNA sequences coding for a chimeric gene containing DNA sequences coding for a polypeptide of interest fused to DNA coding for a polypeptide containing a domain for attachment of biotin, with the DNA for the polypeptide of interest fused to the avidin-binding polypeptide at the 3' end of the DNA coding for the avidin-binding polypeptide.
It is a further object of this invention to provide a process for the production of a hybrid polypeptide of interest by constructing a plasmid for the hybrid polypeptide, transforming that plasmid into a procaryotic or eucaryotic host cell expression system, passing a hybrid polypeptide resulting from said expression system into contact with avidin and harvesting the resulting avidin-bound hybrid polypeptide of interest.
Other objects and advantages will become apparent from the following more complete description and claims.